Agenda Setting - Teorías sobre la Opinión Pública

2.5. Teorías sobre la Opinión Pública

2.5.4. Agenda Setting

972

Figure 1. Post-‐natal brown and white adipose tissue growth requires Rictor 973

(A) Growth curve of control and RictorMyf5cKO_{mice
(n=13;
bars
represent
mean
±
SEM;
t-‐}

974

test; *p<0.05, **p<0.01, ***p<0.001). 975

(B) (Left) Mass of BATs as a percent of total body weight (6-‐wks) (n=19-‐21; bars represent 976

mean ± SEM; t-‐test; ***p<0.001) and (Right) representative image of control and mutant 977

iBAT (6-‐wks). 978

bars represent mean ± SEM; t-‐test; **p<0.01, ***p<0.001) and (Right) representative image 980

of control and mutant rWAT (6-‐wks). 981

(D) Mass of lean tissues expressed as a percent of total body weight (6-‐wks) (n=15-‐19; bars 982

represent mean ± SEM; t-‐test; ***p<0.001). 983

(E) Western blots of lysates prepared from the indicated tissues (6-‐wks). 984

(F) Tissue mass expressed as average weight (mg) at 6-‐wks and 6-‐mos of age (n= 14-‐21 for 985

6-‐wks mice; n=7 for 6-‐mos mice; bars represent mean ± SEM; t-‐test; ***p<0.001). 986

See also Figure S6. 1052

1053

Figure 7. RictorMyf5cKO_{mice
exempt
from
thermal
stress
and
consuming
a
high
fat
diet} 1054

are resistant to obesity and metabolic disease 1055

(A) Weekly weight gain of control and RictorMyf5cKO_{mice
during
12-‐weeks
of
each
normal}

1056

chow diet (chow) or high fat diet (HFD) (n=8 for control and n=12 for KO in chow; n=10 for 1057

both genotypes on HFD; bars represent mean ± SEM; t-‐test; *p<0.05) The control mice 1058

initially weighed 21.63±0.812g in the chow group and 21.24±0.621 in the HFD group; The 1059

RictorMyf5cKO_{mice
initially
weighted
19.42±0.305g
in
the
chow
group
and
19.32±0.348
in}

1060

the HFD group. 1061

(B) Total energy intake (MJ) during the feeding regimen described in (A). Control mice 1062

consumed 3.75±0.557g of chow and 2.81±0.120g of HFD; RictorMyf5cKO_{mice
consumed}

1063

3.85±0.237g of chow and 2.95±0.354g of HFD. 1064

food consumed (n=8 for control and n=12 for KO on chow; n=10 for both genotypes on 1066

HFD; bars represent mean ± SEM; two-‐way ANOVA; *p<0.05, ***p<0.001). 1067

(D) Mass (mg) of the indicated tissues collected from control and KO mice after 12 weeks 1068

on chow or HFD. (n=8 for control and n=12 for KO on chow; n=10 for both genotypes on 1069

HFD; bars represent mean ± SEM; two-‐way ANOVA; *p<0.05, ***p<0.001). 1070

(E-‐F) H&E staining of iBAT and pgWAT and Oil red O staining of livers after 12-‐weeks of 1071

eating chow diet (E) or high fat diet (F). 1072

(G) qRT-‐PCR of the indicated brown and white fat genes in iBAT from chow or HFD mice 1073

(n=8 for control and n=12 for KO on chow; n=10 for both genotypes on HFD; bars 1074

represent mean ± SEM; two-‐way ANOVA; *p<0.05, **p<0.01, ***p<0.001; # indicates 1075

significant difference over the control chow group). 1076

(H) qRT-‐PCR of the indicated metabolic genes in iBAT from chow or HFD mice (n=8 for 1077

control and n=12 for KO in chow; n=10 for both genotypes in HFD; bars represent mean ± 1078

SEM; two-‐way ANOVA; *p<0.05, **p<0.01, ***p<0.001; # indicates significant difference 1079

over the control chow group). 1080

(I) Western immunoblot for UCP1 and the indicated control proteins using lysates 1081

prepared from iBAT. 1082

See also Figure S7. 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Supplemental Information

Rictor/mTORC2 Loss in the Myf5-‐lineage Reprograms Brown Fat Metabolism and Protects Mice against Obesity and Metabolic Disease

Chien-‐Min Hung, Camila Martinez Calejman, Juan Sanchez-‐Gurmaches, Huawei Li, Clary B. Clish, Simone Hettmer, Amy J. Wagers, and David A. Guertin

Inventory of Supplemental Information Figure S1, related to Figure 1

Figure S2, related to Figure 2 Figure S3, related to Figure 3 Figure S4, related to Figure 4 Figure S5, related to Figure 5 Figure S6, related to Figure 6 Figure S7, related to Figure 7

Supplemental Information

Rictor/mTORC2 Loss in the Myf5-‐lineage Reprograms Brown Fat Metabolism and Protects Mice against Obesity and Metabolic Disease

Chien-‐Min Hung, Camila Martinez Calejman, Juan Sanchez-‐Gurmaches, Huawei Li, Clary B. Clish, Simone Hettmer, Amy J. Wagers, and David A. Guertin

Figure S1, Related to Figure 1

(A) Transverse sections of E16.5 embryos. Tongue (1), masseter (2), sternohyoid and hyoglossus (3), supraspinatus (4), prevertebral (5), and trapezius muscles (7), and cervical BAT (7), interscapular BAT (8), and subscapular BAT precursors (9) are indicated. Bracket

marks region of hind neck fragility. (B) Enlarged image of supraspinatus muscle

(arrowhead). Ossifying cartilage of the scapula marked with (*). (C) Enlarged image of prevertebral muscles of the neck (arrowhead). Carotid artery marked with (*). (D)

Enlarged image of trapezius muscle (closed arrowhead) and cervical BAT precursors (open arrowhead). (E) Western blots of satellite cell lysates from control (CT) and RictorMyf5cKO

conditional knockout (cKO) mice. (F) Differentiated satellite cells stained with myosin heavy chain antibody. (G) Quantification of nuclei number in individual differentiated satellite cells. (H) H&E images of tibialis anterior (TA) muscle 1 day after PBS or

cardiotoxin injection in control mice (see also supplementary methods). (I) Mice deleted for Raptor or Rictor specifically in satellite cells with Pax7-‐CreER were subjected to an acute cardiotoxin injury assay. Mice also carried the Rosa26-‐LacZ reporter to follow the deleted cells. H&E images and corresponding images for LacZ staining of TA muscle 10 days after cardiotoxin injection. Regenerated muscle cells in the control and Rictor KO are indicated by the centrally localized nuclei in H&E stained sections. No regenerated cells are detectable in the Raptor KO. (J-‐K) Total body weight (J) and average iBAT weight (K) at postnatal day 1 (n=6; bars represent mean ± SEM; t-‐test; ***p<0.001).

Figure S2, related to Figure 2.

(A) Nuclei density per mm2_{of
iBAT
(6-‐wks)
(n=4;
bars
represent
mean
±
SEM;
t-‐test;}

***p<0.001). (B) Qunatification of genomic DNA from iBAT (6-‐wks) (n=8; bars represent mean ± SEM; t-‐test; *p<0.05). (C) H&E stains (40x) of the quadricep (Quad) muscle and posterior subcutaneous (psWAT) and perigonadal (pgWAT) white adipose tissue (6-‐wks). (D) Representative images of mTFP and mGFP labeled iBAT, psWAT and pgWAT

adipocytes. Note adipocytes are homogenously mGFP+_{and
smaller
in
the
iBAT
consistent}

with homogeneous Rictor loss in this tissue. (E) Top—UCP1 immunohistochemistry stains of iBAT and CL-‐316243 treated psWAT (20x). Botton—asWAT (20x and 40x) from control and RictorMyf5cKO_mice.

Figure S3, related to Figure 3.

(A) Western blots of the indicated total and phospho-‐proteins using lystates prepared from the iBAT of 8-‐week-‐old mice. Overnight fasted mice were i.p. injected with PBS or 150U/Kg insulin and tissues were collected 15 minutes post injection.

Figure S4, related to Figure 4.

(A) Clustering heat map for mitochondrial genes involved in energy metabolism qRT-‐PCR array (n=4). (B) Metabolic cage analysis of 6wk-‐old mice under normal housing

temperature (22°C): Top Left—whole body oxygen consumption; Top right—whole body oxygen consumption normalized to body weight; Botton—food intake, physical activity, respiratory exchange ratio (RER) and energy expenditure. (n=6) (C) Glucose uptake by

18_{FDG
PET-‐CT
(n=6;
bars
represent
mean
±
SEM;
t-‐test;
*p<0.05).

(D)
Metabolite
profiling}

was performed on 6-‐week control and RictorMyf5cKO_{iBAT.

Note
the
high
levels
of
IMP,
a}

deamination product of AMP. AMP is formed by the adenylate kinase reaction, which produces ATP (2ADP = AMP + ATP). During metabolic stress or following treatment with chemical uncouplers, AMP is deaminated to IMP to ensure ongoing adenylate kinase activity and ATP production in order to maintain energy balance (Balcke et al., 2011).

Figure S5, related to Figure 5.

(A) Inducible knockout differentiation protocol for comparing RictoriKO _{to
isogenic
control}

cells. Brown adipocyte precursors (bAPCs) were split from the same original dish into two dishes, one of which received vehicle (EtOH), the other 4-‐hydroxy-‐tamoxifem (4-‐OHT). After 3 days of treatment to induce deletion, cells were passed one time and then differentiated according to a standard 10-‐day brown adipocyte induction protocol

(described in Experimental Procedures). (B) Western immunoblots showing time course following induced Rictor deletion in bAPCs with 4-‐OHT compared to vehicle (EtOH) treated isogenic controls. (C) qRT-‐PCR of mRNA levels for the indicated differentiation-‐related genes (n=3; bars represent mean ± SEM; t-‐test; *p<0.05, ***p<0.001). (D) Left—Oil Red O staining of control and RictorMyf5cKO_{bAPCs
after
differentiation.
Right—Western}

immunoblots showing indicated differentiation markers.

Figure S6, related to Figure 6.

(A) Western immunoblots of undifferentiated control and RictoriKO_{bAPCs
stably
expressing}

the indicated recombinant constructs. Cells were treated with fresh culture media before harvesting. (B) Western immunoblots of AKT1 and AKT2 protein expression at the indicated days during differentiation of wild type bAPCs. (C) Oil Red O staining of

differentiated Akt1 and Akt2 conditional knockout and control bAPCs. The knockout cells were generated from Myf5-‐cre;Akt1fl/fl _{or
Myf5-‐cre;Akt2}fl/fl _{mice
and
the
control
cells
are}

from their Cre-‐negative littermates. (D) Western immunoblots of lysates prepared from differentiated cells in (C). (E) Western immunoblots of lysates generated from AKT isoform-‐specific immunoprecipitation experiments using control or RictoriKO

undifferentiated bAPCs. Immunoblots of the whole cell lystates (WCL) are shown to the left. (F) Western immunoblots of lysates generated from AKT isoform-‐specific

Figure S7, related to Figure 7.

(A) Mass (mg) of the indicated tissues collected from control and RictorMyf5cKO_{mice
living
at}

thermoneutrality (30°C) following 12-‐weeks of eating chow or HFD. (n=8 for control and n=12 for KO in chow; n=10 for both genotypes in HFD; bars represent mean ± SEM; two-‐ way ANOVA; *p<0.05, ***p<0.001) (B) Glucose tolerance test of control and RictorMyf5cKO

during the 11th week of the 12-‐week experiment. (C) qRT-‐PCR of the indicated brown and white fat genes in iBAT from control and RictorMyf5cKO_{mice
eating
chow
diet
and
living
at}

thermoneutrality (n=8 for control and n=12 for KO in chow; bars represent mean ± SEM; two-‐way ANOVA; *p<0.05, **p<0.01, ***p<0.001). The expression level of each gene is normalized to the corresponding gene level in iBAT from age-‐matched control mice eating chow but living at the standard housing temperature (22°C). (D) qRT-‐PCR of the indicated lipogenesis genes in iBAT from chow or HFD mice (n=8 for control and n=12 for KO in chow; n=10 for both genotypes in HFD; bars represent mean ± SEM; two-‐way ANOVA; *p<0.05, **p<0.01, ***p<0.001; # indicates significant difference over the control chow group). (E) Representative H&E images (n=4) of control and KO mice fed with HFD at thermoneutrality for 20 weeks.

Supplemental Experimental Procedures

Embryo analysis

Timed matings were performed and embryos were dissected at the indicated days.

Embryos were fixed overnight in paraformaldehyde, paraffin embedded, and processed for histological analysis according to conventional methods.

Mice

Mice were kept on a daily 12 h light/dark cycle and fed a normal chow diet (Prolab® Isopro® RMH 3000) from LabDiet ad libitum at 22°C (except thermoneutrality studies). All animal experiments were approved by the University of Massachusetts Medical school animal care and use committee.

Satellite cells isolation and in vitro differentiation

Limb muscle including triceps surae (TS), tibialis anterior (TA), quadriceps and triceps were dissected and minced from 6 to 8-‐wks mice. Isolated interstitial and myofiber-‐ associated cells were passed through 70μm nylon mesh and centrifuged at 1200 rpm. Red blood cells were removed from preparations by incubation with RBC lysis buffer (0.15 M ammonium chloride, 0.01 M potassium bicarbonate) on ice for 3 minutes. Antibody staining was performed for 20 min on ice in Hank’s balanced salt solution supplemented with 2% FCS and 2 mM EDTA. After staining cells were filtered through a 35-‐µm cell-‐ strainer capped tube to ensure single cell suspension. Sorting was performed immediately after filtration using a FACS Aria II cell sorter equipped with FASCDiva software. Cells were initially selected by size and shape and only live (PI-‐, calcein blue+) singlets were gated for further analysis of surface markers. Finally an enriched pool of cells (Sca-‐1-‐, Mac1-‐,

Ter119-‐, CD45-‐, CXCR4+ and β1 intergrin+) were purified and re-‐sorted with the same scheme described above to ensure the purity. Double sorted satellite cells were plated at 4x103_{cells/well
in
collagen/laminin
coated
96-‐well
plates.

Cells
were
maintained
in}

growth media (20% horse serum in F10 media, Invitrogen) and feed with 5ng bovine FGF daily for 5 days. For inducing muscle fiber formation, cells were first transferred into matrigel (BD Biosence)-‐coated chamber slides and grown in growth media with bFGF. 2~3

days later, cells were exposed to differentiation media (2% horse serum in F10 without bFGF). After 2 to 4 days myofiber can be observed and fixed with 4% paraformaldehyde. Myosin heavy chains and DAPI staining were performed as described in

immunofluorescence section.

Muscle regeneration after cardiotoxin injury

To induce Rictor deletion in vivo, Pax7-‐CreERT2_{mice
were
i.p.
injected
with
200μg/g
of}

tamoxifen (dissolved in ethanol first then diluted in corn oil to 10mg/mL) for consecutive 4 days. One day later the mice were anesthetized with 12mg/kg xylazine and 60mg/kg ketamine and 30μL cardiotoxin (10μmol/L from Naja nigricollis, Calbiochem) was directly injected into tibialis anterior muscle. 30μL PBS was given in contralateral TA muscle as control. 1 day and 10 days post injury, TA muscle was removed and muscle regeneration was examined by H&E staining and LacZ staining.

LacZ staining

Adipose tissue depots were fixed in 2% paraformaldehyde, 0.2% glutaraldehyde in PBS for 30 min at room temperature. The tissues were then washed 3 times for 15 min in wash buffer (PBS carrying 2 mM MgCl2 and 0.02% Igepal® CA-‐630). Staining was perform in wash buffer containing X-‐gal (1mg/mL), potassium ferricyanide (5 mM) and potassium ferrocyanide (5 mM) at room temperature for at least 16 h. Next, tissues were further fixed in fixing solution for at least 12 h at room temperature, transferred to ethanol for

dehydratation, then sectioned at 5 µm thicknesses. Sections were counter-‐stained with nuclear fast red dehydrated and mounted using CitosealTM 60 (Thermo Scientific). Lean tissues were snap frozen in isopentane-‐liquid nitrogen in OCT. Sections (10 µm) were stained overnight (X-‐gal (1mg/mL), potassium ferricyanide (5 mM) and potassium ferrocyanide (5 mM), MgCl2 (2 mM) in PBS at 37°C and counter-‐stained with nuclear fast red, dehydrated and mounted.

Tissue harvest and histology

first immersed in RNAlater (Invitrogen) and stored at -‐80°C; otherwise, they were frozen down immediately in liquid nitrogen. For histology, tissue pieces were fixed by 10% formalin. Embedding, sectioning and Hematoxylin & Eosin (HE) staining was done by the UMass Morphology Core.

Immunohistochemistry

Adipose tiss sections were subjected to UCP1 IHC according to (Cohen et al., 2014). Briefly, fat sections were hydrated and antigen retrieval was done by incubating the sections in citrate buffer at 90-‐95°C water for 20 min. After blocking, primary antibody (anti-‐Ucp1 antibody, Abcam #ab10983) was applied overnight at 4°C. Next day, SuperPicture 3rd_Gen

IHC Detection Kit (Novex) was used for detection.

Whole-‐mount confocal microscopy

Indicated brown and white adipose tissues were dissected from 6 week-‐old mice and were mounted with Fluoromount-‐G (Southern Biotech) as described in (Berry and Rodeheffer, 2013). Mounted samples were imaged on a LSM 5 Pascal (Zeiss) point scanner confocal system. 40x objective was used with oil immersion. Background fluorescence was offset by using wild-‐type tissues (no mT/mG allele). GFP was excited at 488 nm and detected from 515 to 565 nm and iBAT form Myf5-‐cre;Rosa26mT/mG mice was used as positive control for GFP signal. TdTomato was excited at 543 nm and detected from 575 to 640 nm and pgWAT from mT/mG mice (without Cre-‐driver) was used as positive control for TdTomato.

Nuclei number and cell size quantification

ImageJ was used to quantify nuclei number in iBAT and cell size (diameter) in rWAT and asWAT. For each individual sample, 4 to 6 images were taken and analyzed. Nuclei density was presented as nuclei number per mm2_.

Total genomic DNA was extracted and purified by using DNeasy Blood & Tissue kit

(Qiagen) according to manufacturer’s instruction. Isolated genomic DNA was quantified by NanoDrop 2000 (Thermo Scientific) spectrophotometer.

Immunofluorescence

Frozen section of interscapular brown adipose tissues were thawed and then fixed with methanol for 15 min at room temperature. The fixed sections were washed with 1mL PBS twice and then were permeabilized and blocked with PBSAT buffer (PBS with 1% BSA and 0.5% Triton X-‐100) for 15 min twice. Primary antibody against mitochondria Cox IV (1:100 dilution, CST #4850) was added to sections for overnight incubation. Slides were washed three times with 1mL PBSAT and incubated with secondary antibody conjugated with Alexa-‐568 or Alexa-‐647 (1:1000 dilution, Invitrogen) for 4 hours. Intensive wash was applied to remove unstained antibodies. DAPI was used to stain nuclei for 5 min and washed away by PBS immediately. The slides were embedded with 5μL mounting media (Prolong Gold, Invitrogen).

Glucose uptake

6-‐week old mice (n=5 per each genotype) were i.p. injected with 18_{F-‐FDG,
364-‐483
uCi}

in100 ul saline, and 30 min later the PET imaging was performed in anesthetized animals (1.2-‐2% isoflurane carried in oxygen) immobilized on a Minerve bed (Bioscan).

Immediately after PET acquisition, each mouse was transferred to the NanoSPECT/CT (Bioscan), for the CT acquisition. The PET images were reconstructed without photon attenuation correction using the PETView program (Philips) with the fully 3D iterative reconstruction algorithm, giving a pixel size of 1 mm. The CT acquisition was performed at standard frame resolution, 45 kVp tube voltage and 500 ms of exposure time. The CT reconstruction was accomplished using In-‐VivoScope 1.37 software (Bioscan). The PET image DICOM files were transferred to the NanoSPECT/CT reconstruction workstation to provide the PET/CT fusion images. Volume-‐of-‐Interest (VOI) analysis of the PET

acquisitions was accomplished with the InVivo-‐ Scope 1.37 software.

Transmission electron microscopy

iBATs (n=3 for each group) were dissected from 5 week-‐old mice and subjected to electron microscopy study done by Core Electron Microscopy Facility, UMass medical school.

Metabolite profiling

Brown fat samples were homogenized in four volumes of water using a TissueLyser II (Qiagen) and profiles of polar metabolites were obtained using LC-‐MS. The polar

metabolite profiling methods were developed using reference standards of each metabolite to determine chromatographic retention times and MS multiple reaction monitoring

transitions, declustering potentials and collision energies. The LC-‐MS methods have been recently described (Townsend et al., 2013). Briefly, negative ionization mode data were acquired using an ACQUITY UPLC (Waters) coupled to a 5500 QTRAP triple quadrupole mass spectrometer (AB SCIEX) running hydrophilic interaction chromatography (HILIC) method. A 30µL aliquot of each homogenate was extracted using 120 µL of 80% methanol (VWR) containing 0.05 ng/µL inosine-‐15N4, 0.05 ng/µL thymine-‐d4, and 0.1 ng/µL

glycocholate-‐d4 as internal standards (Cambridge Isotope Laboratories). The samples were centrifuged (10 min, 9,000 x g, 4ºC) and the supernatants (10 µL) were injected directly onto a 150 x 2.0 mm Luna NH2 column (Phenomenex) that was eluted at a flow rate of 400 µL/min. Initial mobile phase conditions were 10% mobile phase A (20 mM ammonium

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